Antonio Piñeiro

Abnormal cell cycle regulation in primary human uveal melanoma cultures

Uveal malignant melanoma is the most frequent primary intraocular tumor in adult humans. The cellular events leading to neoplasic transformation of normal uveal melanocytes are not well known when compared to other cancers. In this study, we investigated the role of G1 and G1/S regulatory proteins of the cell cycle in human uveal melanoma (UM) primary cell cultures, since these proteins are common targets in tumor development. Further, freshly established and characterized tumor cells are a better model for in vitro studies when compared to cell lines established long ago. Human primary cell cultures from eight different UM were established, as well as one primary culture from rhesus uveal normal melanocytes (UNM). Primary human UM cultures were characterized by a low establishment and growing rate. From four successful cultures, three showed a high expression of cyclin D1, cyclin E, p16INK4A, and p27KIP1 with no variations in cyclin A, cyclin‐dependent kinase 2 (CDK2), and CDK4. Interestingly, in one of the cultured tumors, tumor suppressor protein retinoblastoma (Rb) did not bind E2F despite the fact that Rb was found in its hypophosphorylated form. No mutations in either RB1 or the Rb‐binding pocket of E2F‐1 were detected. Furthermore, we identified seven proteins co‐immunoprecipitating with Rb in this tumor, including Lamin A/C and six proteins not previously reported to bind Rb: Hsc70, high mobility group protein 1 (HMG‐1), hnRPN, glyceraldehyde 3 phosphate dehydrogenase (G3PDH), EF‐1, and EF‐2. Our results indicate that the overexpression of cyclins D1/E and CDKIs p16 and p27, together with a deregulation of the Rb/E2F pathway, may be implicated in the development of human UM.

Serum DJ-1/PARK 7 is a potential biomarker of choroidal nevi transformation.

PURPOSE There is substantial evidence that intraocular melanomas arise from benign nevi in the uveal tract. Previous studies performed by the authors revealed that uveal melanoma cells secrete the oncoprotein DJ-1/PARK7 into the extracellular environment and circulation. The aim of this study was to determine whether circulating DJ-1 serum levels correlate with known clinical risk factors of nevi growth. METHODS Standardized ultrasonography, optical coherence tomography, and eye fundus examinations were used to evaluate the clinical risk factors of nevi growth. These clinical risk factors (including nevi size, distance of margins to the optic disc, detection of acoustic hollowness, presence of ocular symptoms, orange pigment, subretinal fluid, and absence of drusen) were examined in 53 consecutive patients from January 2009 to February 2011. Serum levels of DJ-1/PARK7 in these patients and in healthy age- and sex-matched controls (n = 32) were analyzed using ELISA. RESULTS Within the choroidal nevi group, DJ-1 serum levels were higher in those with symptoms (P < 0.033), with a nevus thickness greater than 1.5 mm (P < 0.001), a large basal diameter greater than 8 mm (P < 0.001), and the presence of acoustic hollowness (P < 0.001), compared to those patients without these risk factors. Similar significant differences were found when these at risk nevi subgroups were compared to healthy persons. CONCLUSIONS Elevated serum levels of DJ-1 are associated with choroidal nevi transformation risk factors. Therefore, DJ-1 appears to be a promising factor for predicting the growth of choroidal nevi and may be a potential biomarker of malignancy.

Relationship between macular bending and foveoschisis in myopic patients.

Purpose To investigate factors associated with myopic foveoschisis and macular bending and to determine how the presence of macular bending affects the development of myopic foveoschisis. Methods In a prospective study of 194 eyes of 105 patients with high myopia, we performed complete ophthalmic examinations, optical coherence tomography (OCT), and A-scan ultrasounds. Patients were divided into three groups according to the OCT results. Group 1 consisted of 25 eyes (17 patients) with myopic foveoschisis; group 2 consisted of 36 eyes (20 patients) with macular bending; and group 3 consisted of 135 eyes (68 patients) without macular bending, foveoschisis, or other diseases. Macular bending was defined as a smooth macular elevation observed upon OCT in patients with pathologic myopia. Age, sex, spherical equivalence, axial length (AXL), and OCT findings were obtained and compared to identify factors that are related to myopic foveoschisis and macular bending. Moreover, using the whole data set, we evaluated and correlated myopic foveoschisis with the presence or absence of macular bending to determine whether this bulge in the macular area influences the development of myopic foveoschisis. Results In group 1, all eyes presented posterior staphyloma and two factors were independently associated with a higher risk of having myopic foveoschisis: internal limiting membrane detachment (p < 0.001) and retinal arteriolar traction (p < 0.001). In group 2, only retinal arteriolar traction (p < 0.009) was independently associated with macular bending. Furthermore, macular bending was significantly correlated as a protective factor against myopic foveoschisis (adjusted odds ratio, 0.116; 95% confidence interval, 0.019 to 0.701; p < 0.019); the AXL of patients with the same spherical equivalence and macular bending was significantly shorter than that of patients without macular bending (p = 0.005). Conclusions Intraocular and extraocular wall factors were associated with myopic traction maculopathy, which plays an important role in its pathogenesis. Moreover, macular bending might be a key factor in preventing myopic foveoschisis by decreasing AXL.

A presentation of iridoschisis with plateau iris: an imaging study.

Iridoschisis is an ocular condition characterised by a separation of the mesodermal layer of the iris in which the anterior layer atrophies. According to histopathological studies, iridoschisis presents with fibrosis and tissue atrophy in the iris stroma that originates from the ectoderm, extends to the posterior pigment epithelium and remains intact. The anterior layer of the iris stroma is separated from the rear and is divided into free strands that protrude into the anterior chamber and may damage the corneal endothelium. The most common ages of onset are the sixth and seventh decades of life and this condition usually manifests at a bilateral and sectoral level in the inferior iris. We present a case of iridoschisis associated with plateau iris, which we studied using optical coherence tomography (OCT) on the anterior segment and ultrasonic biomicroscopy (UBM). The objective of this paper was to describe the characteristics of this disease that were detectable using these techniques.

The bovine vitreous‐derived lipid factor (bVLF) is a powerful inhibitor of retinal pigmented epithelial (hRPE) cell proliferation

Human retinal pigmented epithelial cell (hRPE) proliferation plays a significant role in various proliferative diseases associated to the retina that leads to loss of vision, such as proliferative vitreoretinopathy. In the current study, the role of the bovine vitreous lipid factor (bVLF) in hRPE cell proliferation has been investigated. bVLF is a bioactive lipid isolated from the bovine vitreous body with strong Ca2+‐mobilizing activity in fibroblast. In the first approach, the effects of bVLF on Ca2+‐mobilizing activity were investigated in hRPE. The results showed that bVLF induced, in a dose‐dependent manner, a Ca2+ mobilization from PA‐sensitive intracellular stores [non‐Ins(1,4,5)P3‐sensitive stores], in which extracellular Ca2+ participated. The increase in intracellular Ca2+ was associated with a dose‐dependent inhibiting effect on cell proliferation. At a dose of 10 μg/mL, bVLF caused a 26% or a 44% inhibition in hRPE cell proliferation during the 3‐ or the 6‐day culture periods, respectively. These effects appear to be specific in hRPE cells, since EFGR‐T17 fibroblast cells treated with equivalent amounts of bVLF did not show any inhibiting effects. This inhibitory action was not associated to apoptotic/necrotic processes. Furthermore, bVLF inhibited EGF‐, bFGF‐, IGF‐I‐, PDGF‐, HGF‐ and VEGF‐induced proliferation of the hRPE cells. Moreover, this inhibitory response was also observed in FBS‐induced hRPE cell proliferation. bVLF, at a concentration of 10 μg/mL, induced 16% inhibition of proliferation during a culture period of 3 days. This inhibitory action was greater during the 6‐day culture period, exceeding 40%. With regard to this action, the results showed that bVLF has a potent inhibitory effect on ERK1/2 activation, and plays a key role in the control of hRPE cell proliferation. These observations contribute to the knowledge of inhibitory factors responsible for keeping antiproliferative environment that preserve the RPE‐associated activities in normal states. It advances the interesting possibility that this factor or a factor with characteristics common to bVLF might be involved in the pathogenesis of abnormal proliferative eye processes.

160Thr mutation in the rhodopsin gene associated with retinitis pigmentosa.

Mutations in the rhodopsin gene were studied in 23 unrelated Spanish patients with sporadic retinitis pigmentosa (RP). A codon 160 Thr C→A transition was found in 4 of the 23 patients vs. none of the 159 controls (p < 0.001) suggesting that this mutation may be an informative marker in RP.

Intravitreal injections of anti-VEGF agents and antibiotic prophylaxis for endophthalmitis: A systematic review and meta-analysis

We performed a systematic review and meta-analysis to determine whether the use of local antibiotics is a beneficial prophylactic treatment for endophthalmitis in patients treated with anti-VEGF agents. We searched the MEDLINE and EMBASE databases, and the Cochrane Library over the period January 2007 to December 2016. The search terms used included “Endophthalmitis”, “Antibiotic” and “Intravitreal injection”. Studies in which the patients were treated exclusively with intravitreal injections of anti-VEGF were selected. Eight studies fit the inclusion criteria, which included a total of 276,774 injections; 109,178 (39.45%) were associated with the use of antibiotics and 114,821 (60.55%) were not associated with the use of antibiotics. Our meta-analysis indicated a significant risk for endophthalmitis that was 1.70 times greater with the use of antibiotics than that without antibiotics, with a confidence interval of 1.08 to 2.66 (p = 0.02). A meta-regression indicated that the location (operating rooms versus outpatient clinics) of injection did not have a significant effect on the incidence of endophthalmitis. The prophylactic use of antibiotics when administering anti-VEGF intravitreal injections may contribute to a greater incidence of endophthalmitis. This finding, in addition to reducing costs, would eliminate a treatment that has been shown to be unnecessary and even harmful to patients.

ME20-S as a Potential Biomarker for the Evaluation of Uveal Melanoma.

PURPOSE We previously identified the presence of the melanocyte-specific secreted (ME20-S) glycoprotein in secretomes of uveal melanoma (UM) cultures. The aim of this study was to test for the presence and levels of ME20-S in the serum of patients with choroidal nevi and UM and correlate these levels with individual clinical data. METHODS Serum ME20-S levels were determined by ELISA in 111 patients distributed into four categories (53 choroidal nevi, 30 untreated UM, 11 10-year disease-free [DF] UM, 17 hepatic metastatic UM) and 32 age- and sex-matched controls. ME20-S levels were correlated with individual clinical data. RESULTS The UM and the metastatic groups showed significantly higher levels of serum ME20-S than the other groups (P < 0.001). ME20-S levels in the DF patients did not differ from those in the control group. In addition, log-transformed serum ME20-S levels showed a positive correlation with the thickness of the lesion mass in UM patients (regression coefficient 0.0689, 95% confidence interval 0.0689-0.1123, R2 = 27.1%). CONCLUSIONS Elevated ME20-S serum levels are associated with tumor size and advanced stages of UM while low levels are characteristic of DF patients. ME20-S might be a promising serum marker for UM and useful for monitoring metastatic disease.

Development and application of molecularly imprinted polymer – Mn-doped ZnS quantum dot fluorescent optosensing for cocaine screening in oral fluid and serum

A molecularly imprinted polymer - Mn-doped ZnS quantum dot-based fluorescence probe for cocaine abuse screening has been prepared and applied to complex samples such as serum and oral fluid. The fluorescent sensing material was prepared by anchoring a selective MIP for COC on the surface of polyethylene glycol (PEG) modified Mn-doped ZnS quantum dots (QDs). Simple and low cost methods have thus been optimized for assessing cocaine abuse in serum and oral fluid by monitoring fluorescence quenching when cocaine (COC) is present (optimized operating conditions with 1.5mL of 200mgL-1 MIP-coated QDs solution, pH 5.5, and 15min before fluorescence scanning). The matrix effect was found to be important when analyzing oral fluid and serum, and several strategies based on centrifugation for oral fluid and solid phase extraction (SPE) for serum were explored. Two analytical methods were developed for oral fluid. The first one (direct method) requires a centrifugation step (6°C, 4000rpm, 20min) to avoid the matrix effect, and allows for cocaine determination by using an aqueous calibration (1:20 dilution). The second method was developed for oral fluid sampled by Salivette devices, and also requires a further centrifugation (6°C, 4000rpm, 20min) of the recovered oral fluid. This method, however, requires the standard addition technique (1:20 dilution) because of the existence of the matrix effect. Regarding serum samples, a direct method (serum dilution) was not possible, and an SPE procedure was needed to avoid the matrix effect (use of aqueous calibration). The limits of detection and quantification when using the Salivette method were 0.035mgL-1and 0.117mgL-1, respectively; whereas, 0.015mgL-1 (LOD) and 0.050mgL-1 (LOQ) were obtained for serum.

EVALUATION OF VITRECTOMY AND REIMPLANTATION FOLLOWING LATE DISLOCATION OF THE INTRAOCULAR LENS-CAPSULAR BAG COMPLEX: A 3 Years Follow-up Study.

Objectives: To study the predisposition factors for dislocation of the intraocular lens (IOL)—capsular bag complex and analyze the results of subsequent reimplantation surgery. Methods: The exclusion criteria were complicated cataract surgery and minor dislocations that appeared in the first year after cataract surgery. Thirty-six months of monitoring of the reimplantation surgery was required. The primary measurements of results were factors concerning dislocation, the interval between cataract surgery and dislocation, and postoperative complications. Results: Thirty-six patients with dislocation of the capsular bag and lens were identified. Pseudoexfoliation was mentioned in 17 cases (47.2%) and was the main predisposition factor. The average interval between cataract surgery and dislocation was 11.5 years. The dislocated IOL was replaced by a posterior chamber implant in 38% of cases and an anterior chamber implant in 62%. The average best-corrected visual acuity improved significantly after the reposition surgery (P < 0.01). The average monitoring period after secondary reimplantation was 5.95 years (min. 3.03—max. 8.46). Three patients developed bullous keratopathy, all of them with an anterior chamber IOL implantation. Conclusion: According to our knowledge, this article is the one with the longest monitoring time to date. Pseudoexfoliation was the main risk factor for the dislocation of the IOL. Surgery significantly improved best-corrected visual acuity, and the reimplantation of the IOL in the posterior chamber was associated with less serious complications.